Abstract

Organophosphorus pesticides (OPs) remain a potential concern to human health because of their continuing worldwide use. Thiophosphorus OPs, once bioactivated by cytochromes P450 (P450s), form oxon metabolites, which are potent acetylcholinesterase inhibitors. This study investigated the rate of desulfation (activation) and dearylation (detoxification) of parathion and chlorpyrifos in human liver microsomes. In addition, recombinant human P450s were used to quantify, for the first time, the P450-specific kinetic variables (Km and Vmax) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. CYP1A2, 2B6, 2C9, 2C19, 3A4, 3A5, and 3A7 were found to be active to a widely varying degree in parathion metabolism, whereas all, with the exception of CYP2C9, were also found to be active in chlorpyrifos metabolism. CYP2B6 and CYP2C19 demonstrated low Km and high Vmax values for the metabolism of both model compounds, which supports their role as the primary enzymes that regulate metabolism at low-level human exposures to OPs. With Km and Vmax values of 0.61 μM, 4827 pmol/min/nmol P450 and 0.81 μM, 12,544 pmol/min/nmol for formation of paraoxon and chlorpyrifos-oxon, respectively, CYP2B6 favored the desulfation reaction. CYP2C19 activity favored dearylation with Km and Vmax values of 0.60 μM, 2338 pmol/min/nmol P450 and 1.63 μM, 13,128 pmol/min/nmol for formation of p-nitrophenol and 3,4,5-tricholorpyrindinol, respectively. P450-specific kinetic parameters for OP metabolism will be used with age-dependent hepatic P450 content to enhance PBPK/PD models so that OP exposures can be modeled to protect human health in different age groups.

Organophosphorus pesticides (OPs) are currently the most commonly used pesticides in the world, with uses ranging from commercial and home use to agricultural applications, all for controlling unwanted insect pests (Sultatos, 1994). Bioactivation of thiophosphorus OPs results in formation of oxon metabolites, which bind to cholinesterases: both B-esterases, which are irreversibly bound by OPs, and A-esterases (paraoxonase 1), which are reversibly bound through hydrolyzing the active oxon metabolite (Sultatos, 1994; Ecobichon, 2001). B-esterase members include butyrylcholinesterase, carboxylesterase, and, most importantly, for OP action, acetylcholinesterase. Inactivation of acetylcholinesterase results in the accumulation of acetylcholine, causing overstimulation of cholinergic nerves. (Chanda et al., 1997; Ecobichon, 2001).

Parathion and chlorpyrifos often have been used as model compounds, the research base for parathion spanning decades (for review see Knaak et al., 2004). Although parathion use is banned within the United States, it is still used worldwide because of its potency, effectiveness, and low cost. Chlorpyrifos is available for use in the United States and is found in formulations carrying brand names such as Dursban and Lorsban; it is often the insecticide of choice when it comes to cockroach control. Paraoxon and chlorpyrifos-oxon represent the activated forms of parathion and chlorpyrifos, respectively, whereas O,O-diethyl phosphate and p-nitrophenol (PNP), from parathion, or O,O-diethyl phosphate and 3,4,5-trichloropyrindinol (TCP), from chlorpyrifos, represent the more readily cleared detoxification products. The balance between activation and detoxification of OPs determines their risk to humans.

PBPK/PD models, which use previously published kinetic data for OP metabolism generated from mouse, rat, or human liver microsomes have proven to be unstable, nonreproducible, and under-represent interindividual variability (Knaak et al., 2004). To circumvent this issue, modifying PBPK/PD models to use individual P450 activities combined with their respective hepatic P450 content may prove to be more reproducible and stable. To have reliable modeling, accurate and complete kinetic data need to be generated. Kinetic data (Km and Vmax) on the metabolism of OPs by specific P450s along with P450-specific content (pmol of P450/mg of microsomal protein) will generate a PBPK/PD model that can better adjust for age, sex, genetic polymorphisms, or other factors, which may alter P450 content and activity, which, in turn, contribute directly to the risk OPs pose to individuals.

We report in this study the identification of specific human P450s that mediate parathion and chlorpyrifos metabolism, as well as report, for the first time, their respective Km and Vmax values for activation and detoxification. These P450-specific kinetic parameters can then be linked to specific hepatic P450 content values as a function of age for future inclusion in PBPK/PD models.

Experimental Conditions. OP stock solutions were prepared in methanol and stored at –20°C when not in use. Incubations with either human liver microsomes (0.5 mg of protein/ml final concentration) or recombinant P450s (0.03–0.06 nmol P450 final concentration) were carried out in buffer (100 mM Tris-HCl, and 5 mM MgCl2, 1 mM EDTA, and 50 μM iso-OMPA, pH 7.4 at 37°C) in a final volume of 0.5 ml. Reactions were initiated with the addition of 1 mM NADPH and incubated for 2 min at 37°C. EDTA was included to inhibit A-esterases, whereas iso-OMPA inhibited B-esterases (Reiner et al., 1993). The reaction was quenched with 1 volume of ice-cold methanol with 0.1% phosphoric acid and centrifuged; then, the supernatant was transferred to HPLC vials and capped for analysis. All reactions were replicated (n = 3–4) for statistical purposes.

Studies with individual recombinant human P450s were also conducted to identify specific P450s that biotransform these OPs and generate P450-specific kinetic data to predict the P450s that will play a prominent role in vivo. CYP1A2, 2B6, 2C9, 2C19, 3A4, 3A5, and 3A7 were found to metabolize parathion, whereas no detectable metabolites were produced with CYP1A1, 2D6, or 2E1. Figure 1 illustrates Michaelis-Menten plots for three of the most metabolically active P450s (CYP2B6, 2C19, and 3A4) for chlorpyrifos, although CYP1A2, 3A5, and 3A7 were also found to be capable of limited metabolism of chlorpyrifos. The Hill equation was also used for determining the kinetics of CYP3A4 in Fig. 1 (plot not shown) because more than 1 mol of substrate was bound to the enzyme (n > 1); however, both equations resulted in the same Km and Vmax values.

Figure 1 illustrates that the active P450s varied in both their overall activity and affinity (Vmax and Km) values, but also with regard to which reaction was favored, desulfation or dearylation. Table 1 summarizes the Km, Vmax, and Vmax/Km, or intrinsic clearance (CLint), for parathion and chlorpyrifos. CYP2B6 and CYP2C19 have the greatest affinity for parathion metabolism, having K-m values of less than 1.0 μM for both desulfation and dearylation. Higher Vmax values and slightly higher Km values for parathion are observed for CYP1A2, whereas CYP3A4 has the greatest Vmax, but much higher Km values (>30 μM) for both reactions. CYP2B6 also shows a higher affinity and activity for chlorpyrifos metabolism to chlorpyrifos-oxon, with a Km value of 0.81 μM and Vmax of 12,544 pmol/min/nmol P450. In contrast, CYP2C19 has a high affinity and activity for TCP formation (dearylation), with a Km of 1.63 μM and a Vmax of 13,128 pmol/min/nmol P450. As with parathion, CYP3A4 has a high Vmax for chlorpyrifos metabolism but also a high Km (>27 μM) for both reactions.

Kinetic values are given for the metabolism of parathion and chlorpyrifos by recombinant human P450s. Values represent the mean ± S.E.M. of a n = 3, b n = 4, or c n = 6 experiments.

Discussion

This study investigated the activation and detoxification of parathion and chlorpyrifos, two model OPs, in human liver microsomes. In addition, recombinant human P450s were used to quantify the P450-specific kinetic variables for each compound to ultimately refine human PBPK/PD models of OP exposure. These data will be used to modify a PBPK/PD model for human chlorpyrifos exposure that uses kinetic parameters derived from rat hepatic microsomal studies (Timchalk et al., 2002).

Michaelis-Menten plots are shown for chlorpyrifos metabolism by recombinant human CYP2B6 (A and B), CYP2C19 (C and D), or CYP3A4 (E and F). Values represent the mean ± S.E.M of three experiments for CYP2B6 and CYP3A4 and four experiments for CYP2C19.

Although several studies have assessed the in vitro metabolism of OPs by human liver microsomes, the kinetic values may vary widely because of the marked variability of P450 content and activity in procured human specimens and differences in incubation conditions and analytical methodology (for review, see Knaak et al., 2004). Differences in experimental methods include detection of only oxon metabolites, the use of excessive substrate concentrations (supersaturated conditions), and nonphysiological pH ranges. Complications and extensive variability in the procurement of human liver specimens for in vitro studies are a major concern. Often it is not possible to limit warm-ischemic time in liver tissue obtained from organ donors. It is necessary to limit warm-ischemic time before proper cryostorage to have accurate measures of enzyme activity. Furthermore, exposure to drugs and other substances that modulate enzyme activity is often not controlled for in specimens obtained from tissue donors. Thus, there remains considerable uncertainty regarding whether variability in metabolism kinetics reflects true age and genetic variability or is an artifact of human liver specimen procurement and storage. The metabolism of selected OPs has also been assessed using specific recombinant human P450s; however, these earlier studies did not report kinetic parameters (Km, Vmax) for all reactions (Sams et al., 2000, 2004; Tang et al., 2001; Mutch et al., 2003).

In the present study, pooled human liver microsomes demonstrated the ability to metabolize both parathion and chlorpyrifos. Kinetic parameters for the two model OPs varied, as did the ratio of dearylation/desulfation products. Although the results represent an average, the Km and Vmax values using nine substrate concentrations, the results from pooled microsomes did not estimate the interindividual variability that occurs within a population.

Interindividual variability in hepatic metabolism was illustrated in reactions conducted with characterized single-donor human liver microsomes. Marked differences in Km and Vmax values for OP metabolism exist and may be due to variable levels of various P450s within a given microsomal sample from a given donor liver. The higher Km values for specimen SD112 and the high Km values for CYP3A4 (Table 1) suggest that CYP3A4 is playing a primary role in the metabolism of the model OPs by this sample of human liver microsomes. The relative activities of CYP1A2, CYP2B6, and CYP2C19 were lower, whereas CYP3A4 activity was higher in SD112 compared with SD101, supporting the dominant role of CYP3A4 in the metabolism of the two model OPs in SD112.

To better address the limitations of using human liver microsomes for establishing kinetic values, OP metabolism was investigated with recombinant human P450s (summarized in Table 1). As expected, since each P450 isozyme has substrate binding sites that differ, each enzyme demonstrated different Km and Vmax values along with different ratios of product formation. Although CYP3A4 showed the greatest Vmax value for parathion, this enzyme's lack of specificity resulted in the highest Km value, which minimizes the role of CYP3A4 in metabolism at lower OP exposures. CYP2B6 and 2C19 have the lowest Km values for parathion and chlorpyrifos, supporting the major role these forms play in metabolism at low-level real-world exposures.

Due to the low Km values of CYP2B6, its activity is of interest. CYP2B6 activity can be expressed through its Vmax/Km values of 7.87, 2.45, 15.56, and 0.74 for the formation of paraoxon, PNP, chlorpyrifos-oxon, and TCP, respectively (Table 1). Thus, CYP2B6 has greater activity in the formation of the oxon and, thus, may be more important in assessing risk because it preferentially forms the toxic compound over the direct detoxification of the OPs at low level exposures. Conversely, CYP2C19 has the highest Vmax/Km in the formation of PNP and TCP, and thus plays a prominent role in the dearylation (detoxification) reaction.

Current PBPK/PD models for OPs utilize kinetic values from rat liver microsomal metabolism studies that do not reflect human enzymes. The building of kinetic models that use human P450-specific kinetic parameters and specific hepatic P450 content should prove to be more accurate and more easily modified to address factors such as gender, age, or polymorphisms, which may affect P450 protein expression and activity. Human hepatic P450 levels are known to vary across different age groups (Tateishi et al., 1997). In addition, polymorphism in CYP2B6 and 2C19 is known to alter protein expression and/or activity, and, thus, these P450s could potentially serve as biomarkers of susceptibility to these agents. In current PBPK/PD models, which vary parameters based on body weight, infants are treated as small adults, and these models may not be accurate or protective of the most sensitive segment of the population. As OPs are the most widely used class of pesticides in the world, safe levels of exposure need to be set to protect the most sensitive individuals. Determining human P450-specific kinetic parameters for OP metabolism is the first step in constructing more refined models, which will include age-dependent P450 content to better assess risk associated with OP exposure in infants, children, or other sensitive subgroups.